A METHOD FOR THE ANALYSIS OF NATURAL AND SYNTHETIC FOLATE IN FOODS

Authors: Doherty, Robert; BEECHER, GARY - RETIRED-1235-15-00

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2002
Publication Date: 1/1/2003
Citation: Doherty, R.F., Beecher, G.R. 2003. A method for the analysis of natural and synthetic folate in foods. Journal of Agricultural and Food Chemistry. 51:354-361.

Interpretive Summary: Food folates are essential for life and more recently have been associated with increase risk of several chronic diseases. In the U.S., these observations have led to the addition of folic acid to several foods (food fortification). In addition, recommendations for food folate intakes have applied biological activity factors for each of the forms of folates. There currently is unavailable a robust, instrumental procedure that will distinguish between naturally occurring food folates and synthetic folic acid. The proposed procedure in this publication is unique in that it uses `off-the-shelf¿ supplies and instrumentation, and was developed with `normal¿ corporate laboratory schedules in mind. This method was developed around the availability of rat plasma for the source of conjugase as part of the tri-enzyme food digestion and deconjugation system. A high capacity, solid-phase extraction (SPE) column was identified and conditions developed which gave quantitative recovery of 5-methyltetrahydrofolate (MTHF) and folic acid (FA), the most prominent folates in the food supply. The various forms of folates were separated on a C-18 high performance liquid chromatography (HPLC) column which is resistant to degradation at low pH. As a result, the mobile phase was simplified to a gradient of low-pH buffer and acetonitrile. Both MTHF and FA were quantified and qualitatively identified with UV detection. However, detection sensitivity, in the absence of spectral identification, was increased by the introduction of fluorescent detection of MTHF, and UV induced photolysis followed by fluorescent quantification of FA. The application of the system was verified by the analysis of several certified reference materials and foods, and comparing results with certified values and/or total folate values as determined by microbiological assay.

Technical Abstract: The essentiality of dietary folates for human beings has been known for many years. Over the shorter term, biological activities associated with several human maladies and the attenuation of biomarkers for several chronic diseases also has been assigned to folates. In the U.S., these observations have led to the addition of folic acid to several foods and food ingredients (food fortification) and to dietary recommendations that assign biological activity to each of the forms of folate in the food supply. There currently is unavailable a robust, instrumental procedure that will distinguish between naturally occurring food folates and synthetic folic acid as part of the routine analysis of foods. The procedure proposed in this publication is unique in that it uses >off-the-shelf= supplies and instrumentation, to the extent possible, and was developed with >normal= corporate work schedules in mind. This method takes advantage of the tri-enzyme food digestion and folate deconjugation steps, but was optimized with a commercially available rat plasma as the source of conjugase. A high capacity styrene-divinylbenzene based solid-phase extraction (SPE) column was identified, and conditions developed for quantitative recovery of 5-methyltetrahydrofolate (MTHF) and folic acid (FA) with it. The various forms of food folates are separated on a C-18 high performance liquid chromatography (HPLC) column which is resistant to degradation at low pH. As a result, the mobile phase was simplified to a gradient of low-pH phosphate buffer (pH 2.2) and acetonitrile. Although FA does not exhibit fluorescence, a UV-induced photolysis system was added, which is controlled by the HPLC system, so that an appropriate segment of the HPLC column effluent is subjected to photolytic conditions and, thereby, FA can be measured as a fluorescent product. The application of the system was verified by analyzing several certified reference materials and foods, and comparing results with certified values and/or total folate values as determined by microbiological assay.